The present invention relates to an enzyme capable of hydrolysing the 1-acyl group of a phospholipid, that is a phospholipase A1, as well as to processes for the production and to the use of such an enzyme.
Phospholipases are enzymes which act on phospholipids: they are selective enzymes which are classified according to their site of action in the phospholipid molecule. Thus, a Phospholipase A1 hydrolyzes the 1-acyl group of a phospholipid, i.e. it hydrolyzes the bond between the fatty acid and the glycerine residue at the 1-position of the phospholipid. A Phospholipase A2 hydrolyzes the 2-acyl, or central acyl, group and Phospholipases C and D, which are also known as phosphodiesterases, cleave on the two sides of the phosphodiester linkage.
The hydrolysis of a phospholipid by a phospholipase results in the production of a so-called "lysophospholipid". Selective hydrolysis of a phospholipid substrate with a Phospholipase A1 produces a 2-acyl lysophospholipid and selective hydrolysis of a phospholipid with a Phospholipase A2 results in the production of a 1-acyl lysophospholipid.
Although phospholipids are used industrially, lysophospholipids have been shown to be more suitable in certain respects for industrial application. Thus, lysophospholipids show an increased solubility in water, thereby giving them enhanced emulsification properties in oil/water emulsions and an ability to form emulsions which are more stable to changing pH conditions, for example which are stable under acid conditions, and to changing temperatures. Furthermore, the ability of the lysophospholipid to form an emulsion is not reduced by the presence of ions, such as magnesium or calcium ions.
These superior properties of the lysophospholipids means that they are particularly suited for use in many industrial applications, such as in food technology, or in the cosmetics and pharmaceutical industries. It has been shown, furthermore, that the conversion of a phospholipid to a lysophospholipid in phospholipid containing substances, such as food products, generally leads to an improvement of certain of the properties of those substances. Thus, for example, a dough formed from wheat flour which contains lysophospholipids is less sticky than a dough in which the wheat flour does not contain any lysophospholipids. However, it is not yet clear whether these improvements, such as that to the dough, are attributable to the presence of the lysophospholipids alone, or to a synergistic reaction of the lysophospholipids with other substances in the product.
Lecithin is a typical phospholipid widely used in industry, for example in food technology, in animal feed products and in pharmaceutical preparations. Lecithin is a surface active agent, it has antioxidant activity and physiological activating action, as well as other properties. However, an emulsion containing lecithin is less stable than emulsions containing other surface active agents, and this is particularly demonstrated in food technology. Enzymatic hydrolysis of a phospholipid, such as lecithin, to convert at least part of it to a lysophospholipid, has been shown to improve these properties, and thus to enhance the value of the resulting lecithin derivative.
Enzymatic hydrolysis of a phospholipid, using a phospholipase isolated from a micro-organism, is known. Such hydrolysis using a Phospholipase A is described in, for example, Japanese Unexamined Patent Publication No. Sho-58-212783, and the hydrolysis using a lipase is described in Japanese Unexamined Patent Publication No, Sho-63-42691. Furthermore, the enzyme Taka-Diastase.TM., which was isolated from the so-called "enzyme treasure chest" species of Aspergillus, A. oryzae [Biochem. Z., 261 (1933) 275], has demonstrated a lipase activity which is capable of hydrolysing a phospholipid. The most commonly used phospholipase in the industrial hydrolysis of phospholipids is, however, pancreatin, an enzyme which is prepared from the pancreas of pigs and which demonstrates the activity of a Phospholipase A2.
The enzymes isolated from micro-organisms have been shown to have less activity than porcine pancreatic phospholipase A2. Other enzymes, such as the lipases, have a lower substrate specificity as well as additional disadvantages, including a poor yield of lysophospholipid and a lower quality of lysophospholipid final product, due to the presence of by-products. Although the generic term "lipase" may appear to include phospholipases, in fact, the enzymatic activities are distinct, particularly in that the phospholipases are more selective in their site of hydrolysis than the lipases. It is furthermore apparent that some enzymes which demonstrate the activity of a phospholipase also demonstrate a separate activity which is classified as the activity of a lipase. This is more clearly demonstrated in the Examples to the present application.
Although pancreatin has better properties than those prior art enzymes isolated from micro-organisms, hydrolysis of a phospholipid using pancreatin has many disadvantages.
Firstly, it is necessary to make continual adjustments to the pH of the reaction mixture during hydrolysis of a phospholipid substrate with porcine pancreatic Phospholipase A2. The optimum pH for activity of pancreatin is in the range from neutral to weakly alkaline. During the hydrolysis reaction, however, the release of free fatty acids causes the pH to drop, that is it increases the acidity of the reaction mixture, so that, unless counter action is taken, the mixture may become acidic, and therefore outside the optimum pH for activity of the enzyme. The pH of the reaction must, therefore, be monitored carefully throughout the reaction, and it must be adjusted whenever necessary. An additional problem associated with this constant adjustment of the p is the resulting need to remove from the final product at the end of the reaction those substances which were added to adjust the pH during the reaction.
A second problem is that pancreatin is very stable, both to heat treatment and to organic solvents, and it is therefore very difficult to de-activate the enzyme. This means that removal of residual Phospholipase A2 from the hydrolysis mixture at the end of the reaction is very difficult. The presence of residual Phospholipase A2 in the final product, in combination with any original phospholipid substrate not hydrolyzed during the reaction, leaves a potential for further reaction of the residual enzyme on the remaining substrate. The continuing activity of the phospholipase in the product results in the release of free fatty acids into the final product, resulting in a progressive change in the properties of the product. This clearly may decrease the value of the final product.
Traditionally heat treatment has been used in processes involving the use of enzymes to de-activate the residual enzyme. However, porcine pancreatic Phospholipase A2 is not sufficiently de-activated by heat treatment, and even treatment of the enzyme at a temperature of 95.degree. C. for 30 minutes will not sufficiently de-activate the residual enzyme. The use of a higher temperature is impossible in view of the sensitivity of the phospholipid and free fatty acids to heat; these two substances are damaged or broken down at temperatures of around 120.degree. C.
Various solutions to these two major problems have been proposed. Thus, solvent fractionation has been used to remove substances added in order to adjust the pH, as described in U.S. Pat. No. 3,652,397. Alternatively, the enzymatic reaction has been carried out in a non-ionic, non-polar organic solvent, in order to reduce the pH problem [Japanese Unexamined Patent publication No. Hei-3-98590]. A further proposal to overcome the pH problem is described in Japanese Unexamined Patent Publication No. Sho-62-14790 and in Japanese Patent Publication No. Hei-4-81431, which demand that a compound is added to the reaction which reacts with any free fatty acid in the reaction mixture to form a water insoluble metallic soap. The problem of de-activation of the residual enzyme has been addressed by a variety of methods, for example: by a combination of solvent fractionation, using a variety of solvents, and column chromatography, using silica gel, for example; or by drying the phospholipid after treatment with a phospholipase A2, followed by fractionation using a polar solvent [Japanese Unexamined Patent Publication No. Sho-62-262998]. A further method, described in Japanese Unexamined Patent Publication No. Sho-63-233750, involves the treatment of the Phospholipase A2 in the reaction product with a protease, followed by de-activation of the protease by heat treatment.
The solutions so far proposed to solve the problems associated with the use of porcine pancreatic Phospholipase A2 are, however, time consuming, costly and intricate. They may also result in a lower yield and affect the safety of the final product.
There remains, therefore, a need for a method for the production of lysophospholipids which can be used with confidence in many commercial and industrial applications.
The present invention provides a solution to the many problems identified above by providing a novel enzyme preparation which hydrolyzes a phospholipid to produce a lysophospholipid, which enzyme preparation is not dramatically affected by pH, and which is readily de-activated at temperatures which do not have a deleterious effect on the phospholipid or fatty acids in the reaction mixture, which enzyme preparation also has other valuable properties.